Microscale devices and reactions in microscale devices

ABSTRACT

The movement and mixing of microdroplets through microchannels is described employing silicon-based microscale devices, comprising microdroplet transport channels, reaction regions, electrophoresis modules, and radiation detectors. The discrete droplets are differentially heated and propelled through etched channels. Electronic components are fabricated on the same substrate material, allowing sensors and controlling circuitry to be incorporated in the same device.

FIELD OF THE INVENTION

The present invention relates to microfabrication of microscale devicesand reactions in microscale devices, and in particular, movement ofbiological samples in microdroplets through microchannels to initiatebiological reactions.

BACKGROUND

The complexity and power of biological reactions has increaseddramatically over the last thirty years. The initial observations of the"hybridization" process, i.e., the ability of two polymers of nucleicacid containing complementary sequences to find each other and annealthrough base pairing interaction, by Marmur and Lane,Proc.Nat.Acad.Sci., U.S.A. 46, 453 (1960) and Doty, et al.Proc.Nat.Acad.Sci., U.S.A. 46, 461 (1960), have been followed by therefinement of this process into an essential tool of modern biology.

Initial hybridization studies, such as those performed by Hayashi, etal., Proc.Nat.Acad.Sci., U.S.A. 50, 664 (1963), were formed in solution.Further development led to the immobilization of the target DNA or RNAon solid supports. With the discovery of specific restrictionendonucleases by Smith and Wilcox, J.Mol.Biol. 51, 379 (1970), it becamepossible to isolate discrete fragments of DNA. Utilization ofimmobilization techniques, such as those described by Southern,J.Mol.Biol. 98, 503 (1975), in combination with restriction enzymes, hasallowed for the identification by hybridization of singly copy genesamong a mass of fractionated, genomic DNA.

In 1977, two methods for DNA sequencing were reported. These were thechemical degradation method of Maxam and Gilbert (1977 Proc. Natl. AcadSci. USA 74:560) and the enzymatic method of Sanger et al. (1977, Proc.Natl. Acad Sci. USA 74:5463). Both methods generate populations ofradiolabeled oligonucleotides which begin at a fixed point and terminaterandomly at a fixed residue or type of residue. These populations areresolved on polyacrylamide gels which allow the discrimination betweenoligonucleotides that differ in length by as little as one nucleotide.

The Maxam and Gilbert method utilizes a fragment of DNA radiolabeled atone end which is partially cleaved in five separate chemical reactions,each of which is specific for a particular base or type of base. Theproducts of these chemical reactions are five populations of labelledmolecules that extend from the labeled end to the site of chemicalcleavage. This method has remained relatively unchanged since itsinitial development. This method works best for DNA sequences that lieless than 250 nucleotides from the labeled end.

In contrast, the Sanger method is capable of sequencing greater than 500nucleotides in a single set of reactions. The Sanger method is anenzymatic reaction that utilizes chain-terminating dideoxynucleotides(ddNTPs). ddNTPs are chain-terminating because they lack a 3'-hydroxylresidue which prevents formation of a phosphodiester bond with thesucceeding deoxyribonucleotide (dNTP). A small amount of one ddNTP isincluded with the four conventional dNTPs in a polymerization reaction.Polymerization or DNA synthesis is catalyzed by a DNA polymerase. Thereis competition between extension of the chain by incorporation of theconventional dNTPs and termination of the chain by incorporation of addNTP. A short oligonucleotide or primer is annealed to a templatecontaining the DNA to be sequenced. The original protocols requiredsingle-stranded DNA templates. The use of double-stranded templates wasreported later (Chen and Seeburg 1985). The primer provides a 3'hydroxyl group which allows the polymerization of a chain of DNA when apolymerase enzyme and dNTPs are provided.

The original version of the Sanger method utilized the Klenow fragmentof E. coli DNA polymerase. This enzyme has the polymerization and 3' to5' exonuclease activity of the unmodified polymerase but lacks 5' to 3'exonuclease activity. The Klenow fragment has several limitations whenused for enzymatic sequencing. One limitations is the low processivityof the enzyme, which generates a high background of fragments thatterminate by the random dissociation of the enzyme from the templaterather than by the desired termination due to incorporation of a ddNTP.The low processivity also means that the enzyme cannot be used tosequence nucleotides that appear more than ˜250 nucleotides from the 5'end of the primer. A second limitation is that Klenow cannot efficientlyutilize templates which have homopolymer tracts or regions of highsecondary structure. The problems caused by secondary structure in thetemplate can be minimized by running the polymerization reaction at 55°C. (R. Gomer and R. Firtel, "Sequencing homopolymer regions." BethesdaRes. Lab. Focus 7:6 1985).

Improvements to the original Sanger method include the use ofpolymerases other than the Klenow fragment. Reverse transcriptase hasbeen used to sequence templates that have homopolymeric tracts (S.Karanthanasis, "M13 DNA sequencing using reverse transcriptase" BethesdaRes. Lab. Focus 4(3):6 1982; Graham et al., "Direct DNA sequencing usingavian myeloblastosis virus and Moleney murine leukemia virus reversetranscriptase" Bethesda Res. Lab. Focus 8(2):4 1986). Reversetranscriptase is somewhat better than the Klenow enzyme at utilizingtemplates containing homopolymer tracts.

The use of a modified T7 DNA polymerase (Sequenase™) was the mostsignificant improvement to the Sanger method. See Sambrook, J. et al.Molecular Cloning, A Laboratory Manual, 2d Ed. Cold Spring HarborLaboratory Press, New York, 13.7-13.9 and Hunkapiller, M. W. (1991)Curr. Op. Gen. Devl. 1:88-92. Sequenase™ is a chemically-modified T7 DNApolymerase has reduced 3' to 5' exonuclease activity (Tabor et al. 1987,Proc. Natl. Acad. Sci. USA 84:4767). Sequenase™ version 2.0 is agenetically engineered form of the T7 polymerase which completely lacks3' to 5' exonuclease activity. Sequenase™ has a very high processivityand high rate of polymerization. It can efficiently incorporatenucleotide analogs such as dITP and 7-deaza-dGTP which are used toresolve regions of compression in sequencing gels. In regions of DNAcontaining a high G+C content, Hoogsteen bond formation can occur whichleads to compressions in the DNA. These compressions result in aberrantmigration patterns of oligonucleotide strands on sequencing gels.Because these base analogs pair weakly with conventional nucleotides,intrastrand secondary structures are alleviated. In contrast, Klenowdoes not incorporate these analogs as efficiently. The main limitationto the amount of DNA sequence that can be obtained from a single set ofchain-termination reactions using Sequenase™ is the resolving power ofpolyacrylamide gels, not the properties of the enzyme.

The use of Taq DNA polymerase is a more recent addition to theimprovements of the Sanger method (Innis et al 1988, Proc. Natl. Acad.Sci. USA 85:9436). Taq polymerase is a thermostable enzyme which worksefficiently at 70-75° C. The ability to catalyze DNA synthesis atelevated temperature makes Taq polymerase useful for sequencingtemplates which have extensive secondary structures at 37° C. (thestandard temperature used for Klenow and Sequenase™ reactions). Taqpolymerase, like Sequenase™, has a high degree of processivity and likeSequenase 2.0, it lacks 3' to 5' nuclease activity.

Methods were also developed for examining single base changes withoutdirect sequencing. These methods allow for the "scanning" of DNAfragments for the presence of mutations or other sequence variation. Forexample, if a mutation of interest happens to fall within a restrictionrecognition sequence, a change in the pattern of digestion can be usedas a diagnostic tool (e.g., restriction fragment length polymorphism[RFLP] analysis).

With the development of these complex and powerful biologicaltechniques, an ambitious project has been undertaken. This project,called the Human Genome Project (HGP), involves the completecharacterization of the archetypal human genome sequence which comprises3×10⁹ DNA nucleotide base pairs. An implicit goal of the project is therecognition that all humans are greater than 99% identical at the DNAsequence level. The differences between people, however, provide theinformation most relevant to individual health care, including potentialestimates of the risk of disease or the response to a specific medicaltreatment. Upon completion of the HGP, a continuing effort of the humangenetics research community will be the examination of differenceswithin populations and of individual variants from the definedarchetype. While the 15-year effort of the HGP represents a definedquantity of DNA data acquisition, the future demand for DNA informationis tied to individual genetic variation and is, therefore, unlimited.

Current DNA genotyping technologies are adequate for the detailedanalysis of samples that range in number from hundreds to thousands peryear. Genotyping projects on the order of millions of assays, however,are beyond the capabilities of today's laboratories because of thecurrent inefficiencies in (i) liquid handling of reagent and DNAtemplate solutions, (ii) measurement of solution volumes, (iii) mixingof reagent and template, (iv) controlled thermal reaction of the mixedsolutions, (v) sample loading onto an electrophoresis gel, and (vi) DNAproduct detection on size-separating gels. What is needed is methodologythat allows for a high-volume of biological reactions without theseexisting inefficiencies.

SUMMARY OF THE INVENTION

The present invention relates to microfabrication of microscale devicesand reactions in microscale devices, and in particular, movement ofbiological samples in microdroplets through microchannels to initiatebiological reactions. The present invention contemplates microscaledevices, comprising microdroplet transport channels, reaction regions(e.g. chambers), electrophoresis modules, and radiation detectors. In apreferred embodiment, these elements are microfabricated from siliconand glass substrates. The various components are linked (i.e., in liquidcommunication) using a surface-tension-gradient mechanism in whichdiscrete droplets are differentially heated and propelled through etchedchannels. Electronic components are fabricated on the same substratematerial, allowing sensors and controlling circuitry to be incorporatedin the same device. Since all of the components are made usingconventional photolithographic techniques, multi-component devices canbe readily assembled into complex, integrated systems.

It is not intended that the present invention be limited by the natureof the reactions carried out in the microscale device. Reactionsinclude, but are not limited to, chemical and biological reactions.Biological reactions include, but are not limited to sequencing,restriction enzyme digests, RFLP, nucleic acid amplification, and gelelectrophoresis. It is also not intended that the invention be limitedby the particular purpose for carrying out the biological reactions. Inone medical diagnostic application, it may be desirable to differentiatebetween a heterozygotic and homozygotic target and, in the latter case,specifying which homozygote is present. Where a given genetic locusmight code for allele A or allele a, the assay allows for thedifferentiation of an AA from an Aa from an aa pair of alleles. Inanother medical diagnostic application, it may be desirable to simplydetect the presence or absence of specific allelic variants of pathogensin a clinical sample. For example, different species or subspecies ofbacteria may have different susceptibilities to antibiotics; rapididentification of the specific species or subspecies present aidsdiagnosis and allows initiation of appropriate treatment.

The present invention contemplates a method for moving microdroplets,comprising: (a) providing a liquid microdroplet disposed within amicrodroplet transport channel etched in silicon, said channel in liquidcommunication with a reaction region via said transport channel andseparated from a microdroplet flow-directing means by a liquid barrier;and (b) conveying said microdroplet in said transport channel to saidreaction region via said microdroplet flow-directing means. It isintended that the present invention be limited by the particular natureof the microdroplet flow-directing means. In one embodiment, itcomprises a series of aluminum heating elements arrayed along saidtransport channel and the microdroplets are conveyed by differentialheating of the microdroplet by the heating elements.

It has been found empirically that the methods and devices of thepresent invention can be used with success when, prior to the conveyingdescribed above the transport channel (or channels) is treated with ahydrophilicity-enhancing compound. It is not intended that the inventionbe limited by exactly when the treatment takes place. Indeed, there issome flexibility because of the long-life characteristics of someenhancing compounds.

Again, it has been found empirically that there is a need for a liquidbarrier between the liquid in the channels and the electronics of thesilicon chip. A preferred barrier comprises a first silicon oxide layer,a silicon nitride layer, and a second silicon oxide layer.

The present invention further contemplates a method for mergingmicrodroplets comprising: (a) providing first and second liquidmicrodroplets, a liquid microdroplet delivering means, and a device,said device comprising: i) a housing comprised of silicon, ii) first andsecond microdroplet transport channels etched in said silicon andconnecting to form a third transport channel containing a reactionregion, iii) a microdroplet receiving means in liquid communication withsaid reaction region via said transport channels, and iv) microdropletflow-directing means arrayed along said first, second and thirdtransport channels; (b) delivering said first liquid microdroplet viasaid microdroplet delivering means to said first transport channel; (c)delivering said second liquid microdroplet via said microdropletdelivering means to said second transport channel; and (d) conveyingsaid microdroplets in said transport channels to said reaction region insaid third transport channel via said microdroplet flow-directing means,thereby merging said first and second microdroplets to create a mergedmicrodroplet.

In one embodiment, said first microdroplet comprises nucleic acid andsaid second microdroplet comprises a nuclease capable of acting on saidnucleic acid. In this embodiment, it is desirable to enhance the mixingwithin the merged microdroplet. This can be achieved a number of ways.In one embodiment for mixing, after the conveying of step (d), the flowdirection is reversed. It is not intended that the present invention belimited by the nature or number of reversals. If the flow direction ofsaid merged microdroplet is reversed even a single time, this processincreases the mixing of the reactants.

The present invention contemplates a variety of silicon-based,microdroplet transport channel-containing devices. In one embodiment,the device comprises: i) a housing comprised of silicon, ii) amicrodroplet transport channel etched in said silicon, iii) amicrodroplet receiving means in liquid communication with a reactionregion via said transport channels, and iv) a liquid barrier disposedbetween said transport channels and a microdroplet flow-directing means.In one embodiment, the device is assembled in two parts. First, thechannels are etched in any number of configurations. Secondly, thispiece is bonded with a silicon-based chip containing the electronics.This allows for both customization (in the first piece) andstandardization (in the second piece).

DEFINITIONS

The following definitions are provided for the terms used herein:

"Biological reactions" means reactions involving biomolecules such asenzymes (e.g., polymerases, nucleases, etc.) and nucleic acids (both RNAand DNA). Biological samples are those containing biomolecules, suchproteins, lipids, nucleic acids. The sample may be from a microorganism(e.g., bacterial culture) or from an animal, including humans (e.g.blood, urine, etc.). Alternatively, the sample may have been subject topurification (e.g. extraction) or other treatment. Biological reactionsrequire some degree of biocompatability with the device. That is to say,the reactions ideally should not be substantially inhibited by thecharacteristics or nature of the device components.

"Chemical reactions" means reactions involving chemical reactants, suchas inorganic compounds.

"Channels" are pathways through a medium (e.g., silicon) that allow formovement of liquids and gasses. Channels thus can connect othercomponents, i.e., keep components "in liquid communication.""Microdroplet transport channels" are channels configured (in microns)so as to accommodate "microdroplets." While it is not intended that thepresent invention be limited by precise dimensions of the channels orprecise volumes for microdroplets, illustrative ranges for channels andmicrodroplets are as follows: the channels can be between 0.35 and 50 μmin depth (preferably 20 μm) and between 50 and 1000 μm in width(preferably 500 μm), and the volume of the microdroplets can range(calculated from their lengths) between approximately one (1) and (100)nanoliters (more typically between ten and fifty).

"Conveying" means "causing to be moved through" as in the case where amicrodroplet is conveyed through a transport channel to a particularpoint, such as a reaction region. Conveying can be accomplished viaflow-directing means.

"Flow-directing means" is any means by which movement of a microdropletin a particular direction is achieved. A preferred directing meansemploys a surface-tension-gradient mechanism in which discrete dropletsare differentially heated and propelled through etched channels.

"Hydrophilicity-enhancing compounds" are those compounds or preparationsthat enhance the hydrophilicity of a component, such as thehydrophilicity of a transport channel. The definition is functional,rather than structural. For example, Rain-X™ anti-fog is a commerciallyavailable reagent containing glycols and siloxanes in ethyl alcohol.However, the fact that it renders a glass or silicon surface morehydrophilic is more important than the reagent's particular formula.

"Initiating a reaction" means causing a reaction to take place.Reactions can be initiated by any means (e.g., heat, wavelengths oflight, addition of a catalyst, etc.)

"Liquid barrier" or "moisture barrier" is any structure or treatmentprocess on existing structures that prevents short circuits and/ordamage to electronic elements (e.g., prevents the destruction of thealuminum heating elements). In one embodiment of the present invention,the liquid barrier comprises a first silicon oxide layer, a siliconnitride layer, and a second silicon oxide layer.

"Merging" is distinct from "mixing." When a first and secondmicrodroplet is merged to create a merged microdroplet, the liquid mayor may not be mixed. Moreover, the degree of mixing in a mergedmicrodroplet can be enhanced by a variety of techniques contemplated bythe present invention, including by not limited to reversing the flowdirection of the merged microdroplet.

"Nucleic Acid Amplification" involves increasing the concentration ofnucleic acid, and in particular, the concentration of a particular pieceof nucleic acid. A preferred technique is known as the "polymerase chainreaction." Mullis, et al., U.S. Pat. Nos. 4,683,195 and 4,683,202,hereby incorporated by reference, describe a method for increasing theconcentration of a segment of target sequence in a mixture of genomicDNA without cloning or purification. This process for amplifying thetarget sequence consists of introducing a molar excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence. The two primers are complementary to their respective strandsof the double-stranded sequence. The mixture is denatured and thenallowed to hybridize. Following hybridization, the primers are extendedwith polymerase so as to form complementary strands. The steps ofdenaturation, hybridization, and polymerase extension can be repeated asoften as needed to obtain are relatively high concentration of a segmentof the desired target sequence. The length of the segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to by the inventors as the "Polymerase Chain Reaction"(hereinafter PCR). Because the desired segment of the target sequencebecome the dominant sequences (in terms of concentration) in themixture, they are said to be "PCR-amplified."

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an integrated analysis system of the presentinvention.

FIG. 2 shows a two-part approach to construction of a silicon device ofthe present invention.

FIG. 3 is a schematic showing the principle of thermally-induced liquidmicrodroplet motion in a closed channel.

FIG. 4A shows a selected frame of a videotape wherein two microdropletsare at their starting locations in the branches of the Y-channel.

FIG. 4B shows movement by heating the left interface of bothmicrodroplets.

FIG. 4C shows the microdroplets at the intersection.

FIG. 4D shows the merging of the microdroplets to form the combinedmicrodroplet. The open arrowheads in the figure indicate the rearmeniscus and the filled arrowheads the leading meniscus for eachmicrodroplet.

FIG. 5A is a photomicrograph of inlay-process heater elements on thesurface of a silicon wafer.

FIG. 5B is a scanning electron micrograph (SEM) of an inlay-processheater wire in cross section (the arrows indicate the depositedaluminum, silicon dioxide, and silicon nitride layers).

FIG. 5C is a SEM of a channel formed on glass using a wet-etch process,shown in cross section with the etched face of the wafer immediatelyadjacent to the intersection of two channels.

FIG. 6A is a photomicrograph of polyacrylamide gel electrophoresis in awide etched-glass channel.

FIG. 6B is a photomicrograph of a set of four doped-diffusion dioderadiation detector elements fabricated on a silicon wafer.

FIG. 6C is an oscilloscope trace of output from the radiation detectorshowing individual decay events from ³² P-labeled DNA.

FIG. 7 is a photo of gel electrophoresis of PCR reactions whereinpotentially inhibiting components were added directly to the PCR.

DESCRIPTION OF THE INVENTION

The present invention relates to microfabrication and biologicalreactions in microfabricated devices, and in particular, movement andmixing of biological samples in microdroplets through microchannels. Thedescription of the invention involves I) design of microscale devices(comprising microdroplet transport channels, reaction chambers,electrophoresis ports, and radiation detectors) using silicon and glasssubstrates, II) movement of discrete microdroplets using asurface-tension-gradient mechanism in which discrete microdroplets aredifferentially heated and propelled through etched channels, and III)mixing of biological samples for reactions.

I. Design Of MicroScale Devices

Although there are many formats, materials, and size scales forconstructing integrated fluidic systems, the present inventioncontemplates silicon microfabricated devices as a cost-effectivesolution. Silicon is the material used for the construction of computingmicroprocessors and its fabrication technologies have developed at anunprecedented pace over the past 30 years. While this technology wasinitially applied to making microelectronic devices, the same techniquesare currently being used for micromechanical systems.

Continuous flow liquid transport has been described using a microfluidicdevice developed with silicon. See J. Pfahler et al., Sensors andActuators, A21-A23 (1990), pp. 431-434. Pumps have also been described,using external forces to create flow, based on micromachining ofsilicon. See H. T. G. Van Lintel et al., Sensors and Actuators15:153-167 (1988). By contrast, the present invention employs discretedroplet transport in silicon (i.e., in contrast to continuous flow)using internal forces (i.e., in contrast to the use of external forcescreated by pumps).

As a mechanical building material, silicon has well-known fabricationcharacteristics. The economic attraction of silicon devices is thattheir associated micromachining technologies are, essentially,photographic reproduction techniques. In these processes, transparenttemplates or masks containing opaque designs are used to photodefineobjects on the surface of the silicon substrate. The patterns on thetemplates are generated with computer-aided design programs and candelineate structures with line-widths of less than one micron. Once atemplate is generated, it can be used almost indefinitely to produceidentical replicate structures. Consequently, even extremely complexmicromachines can be reproduced in mass quantities and at lowincremental unit cost--provided that all of the components arecompatible with the silicon micromachining process. While othersubstrates, such as glass or quartz, can use photolithographic methodsto construct microfabricated analysis devices, only silicon gives theadded advantage of allowing a large variety of electronic components tobe fabricated within the same structure.

In one embodiment, the present invention contemplates siliconmicromachined components in an integrated analysis system, including theelements identified schematically in FIG. 1. In this proposed format,sample and reagent are injected into the device through entry ports (A)and they are transported as discrete droplets through channels (B) to areaction chamber, such as a thermally controlled reactor where mixingand reactions (e.g., restriction enzyme digestion or nucleic acidamplification) occur (C). The biochemical products are then moved by thesame method to an electrophoresis module (D) where migration data iscollected by a detector (E) and transmitted to a recording instrument(not shown). Importantly, the fluidic and electronic components aredesigned to be fully compatible in function and construction with thebiological reactions and reagents.

In silicon micromachining, a simple technique to form closed channelsinvolves etching an open trough on the surface of a substrate and thenbonding a second, unetched substrate over the open channel. There are awide variety of isotropic and anisotropic etch reagents, either liquidor gaseous, that can produce channels with well-defined side walls anduniform etch depths. Since the paths of the channels are defined by thephoto-process mask, the complexity of channel patterns on the device isvirtually unlimited. Controlled etching can also produce sample entryholes that pass completely through the substrate, resulting in entryports on the outside surface of the device connected to channelstructures.

FIG. 2 shows a two-part approach to construction. Microchannels (100)are made in the silicon substrate (200) and the structure is bonded to aglass substrate (300). The two-part channel construction techniquerequires alignment and bonding processes but is amenable to a variety ofsubstrates and channel profiles. In other words, for manufacturingpurposes, the two-part approach allows for customizing one piece (i.e.,the silicon with channels and reaction formats) and bonding with astandardized (non-customized) second piece, e.g., containing standardelectrical pads (400).

II. Movement Of Discrete MicroDroplets

The present invention describes the controlled movement of liquidsamples in discrete droplets in silicon. Discrete droplet transportinvolves a system using enclosed channels or tubes to transport theliquid to the desired locations (FIG. 1, B). Within the channels,discrete liquid reagent microdroplets can be injected, measured, andmoved between the biochemical analysis components. Discrete dropletmovement has three advantages. First, each sample droplet is separatedfrom all others so that the risk of contamination is reduced. Second, ina uniform channel, the volume of each sample can be determined by merelymeasuring the droplet length. Third, the motion of these droplets can beaccomplished with simple heating (i.e., using internal forces and nomoving parts). Movement is performed using thermal gradients to changethe interfacial tension at the front or back of the droplets and, thus,generate pressure differences across the droplet (FIG. 3). For example,a droplet in a hydrophilic channel can be propelled forward by heatingthe back interface. The local increase in temperature reduces thesurface tension on the back surface of the droplet and, therefore,decreases the interfacial pressure difference. The decreased pressuredifference corresponds to an increase in the local internal pressure onthat end of the droplet (P₁ increases). The two droplet interfaces areno longer in equilibrium, with P₁ greater than P₂, and the pressuredifference propels the droplet forward.

That is to say, forward motion can be maintained by continuing to heatthe droplet at the rear surface with successive heaters along thechannel, while heating the front surface can be used to reverse themotion of the droplet. Applying a voltage to the wire beneath thechannel generates heat under the edge of the droplet. Heating the leftinterface increases the internal pressure on that end of the droplet andforces the entire droplet to the right. The pressure on the interior ofthe droplet can be calculated knowing the atmospheric pressure, P_(atmP)the surface tension, σ, and the dimensions of the channel. For acircular cross-section, the interior pressure, P_(i), is given by P_(i)=P_(atm) -(4σ cos θ)/d where d is the diameter of the channel and θ isthe contact angle. Since σ is a function of temperature (σ=σ_(o) (1-bT)where σ_(o) and b are positive constants and T is the temperature),increasing the temperature on the left end of the droplet decreases thesurface tension and, therefore, increases the internal pressure on thatend. The pressure difference between the two ends then pushes thedroplet towards the direction of lower pressure (i.e., towards theright). The aqueous droplet shown is in a hydrophilic channel (0<θ<90);for a hydrophobic channel (90<θ<180), heating the right edge would makethe droplet move to the right.

Contact angle hysteresis (the contact angle on the advancing edge of thedroplet is larger than the contact angle on the retreating edge)requires a minimum temperature difference before movement will occur.The velocity of the droplet after motion begins can be approximatedusing the equation ν=AEPd² /32 μL where AEP is the pressure difference,μ is the viscosity of the solution, and L is the length of the droplet.The present invention contemplates temperature differences of greaterthan thirty (30) degrees Centigrade to create movement. Experimentsusing temperature sensors arrayed along the entire channel indicate thata differential of approximately 40° C. across the droplet is sufficientto provide motion. In these experiments, the channel cross-section was20 μm×500 μm, and the volume of each of these droplets can be calculatedfrom their lengths and is approximately 100 nanoliters for a 1 cm longdroplet.

III. Mixing Biological Samples In Reactions

Droplet motion (described generally above) is contemplated as one stepin a pathway. The other steps typically involve sample mixing and acontrolled reaction. For example, the integral heaters arrayed along theentire surface of the channel used for droplet motion also allow for aregion of a channel to be used as a thermal reaction chamber. For samplemixing prior to the reaction, a Y-channel device is contemplated (FIG.4A). In such a device, a first droplet containing a first sample (e.g.,nucleic acid) is moved along one channel of the Y-channel device, and asecond droplet containing a second sample (e.g., a restriction digestenzyme in digestion buffer) is moved along the other channel of theY-channel device (FIGS. 4B and 4C).

Following sample merging (FIG. 4D), there is the concern that thecombined samples have not been properly mixed. That is to say, if twosimilar microdroplets enter the single channel in laminar flow at thesame flow rate, they will form an axially uniform droplet but will notbe mixed width-wise. Width-mixing can be accomplished in a number ofways.

First, there is simple diffusion, although, for large DNA molecules, thecharacteristic time for this mixing could be on the order of severalhours or more. Circulation patterns generated inside the droplets duringmovement and heating significantly reduce this time. In this regard, thepresent invention contemplates maintaining the mixture as a heatedmixture (e.g., maintaining the temperature at 65° C. for 10 minutes)using the integral heaters and temperature sensors.

Second, the present invention contemplates mixing by reversing the flowdirection of the mixture over a relatively short distance in thechannel. While a variety of reverse flow approaches are possible, one ortwo direction changes over a distance comprising approximately twodroplet lengths has been found to be adequate.

Finally, there is the mixing approach wherein the mixture is movedagainst or over physical obstacles. For example, the mixture can beeither "crashed" back against merge point of the Y-channel or simplymoved over deliberate imperfections in the channel (i.e., "rollercoaster" mixing).

Successful mixing, of course, can be confirmed by characterization ofthe product(s) from the reaction. Where product is detected, mixing hasbeen at least partially successful. The present invention contemplates,in one embodiment, using electrophoresis to confirm product formation.

DESCRIPTION OF PREFERRED EMBODIMENTS

The description of the preferred embodiments involves: I)microfabrication techniques for manufacture of silicon-based devices;II) channel treatment for optimum flow and reproducibility; and III)component design (particularly the electrophoresis module and theradiation detectors).

I. Microfabrication Of Silicon-Based Devices

As noted previously, silicon has well-known fabrication characteristicsand associated photographic reproduction techniques. The principalmodern method for fabricating semiconductor integrated circuits is theso-called planar process. The planar process relies on the uniquecharacteristics of silicon and comprises a complex sequence ofmanufacturing steps involving deposition, oxidation, photolithography,diffusion and/or ion implantation, and metallization, to fabricate a"layered" integrated circuit device in a silicon substrate. See e.g., W.Miller, U.S. Pat. No. 5,091,328, hereby incorporated by reference.

For example, oxidation of a crystalline silicon substrate results in theformation of a layer of silicon dioxide on the substrate surface.Photolithography can then be used to selectively pattern and etch thesilicon dioxide layer to expose a portion of the underlying substrate.These openings in the silicon dioxide layer allow for the introduction("doping") of ions ("dopant") into defined areas of the underlyingsilicon. The silicon dioxide acts as a mask; that is, doping only occurswhere there are openings. Careful control of the doping process and ofthe type of dopant allows for the creation of localized areas ofdifferent electrical resistivity in the silicon. The particularplacement of acceptor ion-doped (positive free hole, "p") regions anddonor ion-doped (negative free electron, "n") regions in large partdefines the interrelated design of the transistors, resistors,capacitors and other circuit elements on the silicon wafer. Electricalinterconnection and contact to the various p or n regions that make upthe integrated circuit is made by a deposition of a thin film ofconductive material, usually aluminum or polysilicon, thereby finalizingthe design of the integrated circuit.

Of course, the particular fabrication process and sequence used willdepend on the desired characteristics of the device. Today, one canchoose from among a wide variety of devices and circuits to implement adesired digital or analog logic feature.

In a preferred embodiment, channels were prepared on 500 μm thick glasswafers (Dow Corning 7740) using standard aqueous-based etch procedures.The initial glass surface was cleaned and received two layers ofelectron beam evaporated metal (20 nm chromium followed by 50 nm gold).Photoresist Microposit 1813 (Shipley Co.) was applied 4000 rpm, 30seconds; patterned using glass mask 1 and developed. The metal layerswere etched in chromium etchant (Cr-14, Cyantek Inc.) and gold etchant(Gold Etchant TFA, Transene Co.) until the pattern was clearly visibleon the glass surface. The accessible glass was then etched in a solutionof hydrofluoric acid and water (1:1, v/v). Etch rates were estimatedusing test wafers, with the final etch typically giving channel depthsof 20 to 30 μm. For each wafer, the depth of the finished channel wasdetermined using a surface profilometer. The final stripping (PRS-2000,J.T. Baker) removed both the remaining photoresist material and theoverlying metal.

In one embodiment, channels etched on glass in the above-describedmanner, were bonded to the heater-element wafer in a two-partconstruction approach using optical adhesive (SK-9 Lens Bond, SumersLaboratories, Fort Washington, Pa.). The bond was cured under anultraviolet light source (365 nm) for 12 to 24 hours.

Initial device design by the present inventors involved single layers ofsilicon. However, experience showed these to be inadequate to preventshort circuiting due to (necessary) liquid microdroplets within thechannels (see experiments described below). The preferred designinvolves a triple layer of oxides. Such a preferred device capable ofmoving and mixing nanoliter droplets was constructed by bonding a planarsilicon substrate to channels etched in a glass cover. A series of metalheaters was inlaid on the silicon substrate as two parallel lanesmerging into a single lane (a "Y"-shape) (FIG. 5A). The heating elementswere formed by first coating the wafer with a 1.0 μm layer of thermalsilicon dioxide Next, 0.35 μm deep, 5 μm wide grooves were reactive-ionetched (RIE) into the silicon dioxide following the pattern set in anoverlying photoresist. Aluminum was deposited (0.35 μm) across theentire wafer using electron beam evaporation and the metal layer was"lifted-off" from all surfaces having intact photoresist using astripping solution. The metal inlay process gives a relatively planarsurface and provides a uniform base for deposition of asolution-impermeable barrier layer. The barrier layer is made by asequence of three plasma-enhanced chemical vapor depositions (PECVD):1.0 μm silicon oxide (SiO_(x)), 0.25 μm silicon nitride (Si_(x) N_(y)),and 1.0 μm silicon oxide (SiO_(x)) (FIG. 5B). Some heating elements werealso used as resistive temperature sensors.

Heater elements were fabricated as follows. Silicon wafer (p-type,18-221/2-cm, <100>, boron concentration Å 10¹⁵ cm⁻³) was used as asubstrate for growth of SiO₂ thermal oxide (1 μm); photoresist(AZ-5214-E, Hoescht-Celanese) was applied and spun at 3000 rpm, 30seconds. The resist was patterned (metal 1) and developed. Reactive ionetch (RIE, PlasmaTherm, Inc.) was performed to 0.35 μm depth into theSiO₂ layer at the following conditions: CHF₃, 15 sccm (standard cubiccentimeters per minute); CF₄, 15 sccm; 4 mTorr; DC bias voltage of 200V,100 W, 20 minutes. The etch depth was measured by profilometer and 0.35μm metallic aluminum was electron beam deposited. The resist andoverlying metal was lifted off by development using Microposit 1112Aremover in solution (Shipley Co.). The barrier layers consist ofsequentially deposited 1 μm SiO_(x), 0.25 μm Si_(x) N_(y), and 1 μmSiO_(x) using plasma-enhanced chemical vapor deposition (PECVD). RIE wasused to etch contact holes to the metal layer using a second mask (CHF₃,15 sccm; CF₄, 15 sccm; 4 mTorr; and DC bias voltage of 200V, 100 W, 120minutes).

As shown in FIG. 5, the elements are arrayed as two parallel lanes, each500 μm wide, merging into one lane. The individual heaters consist ofpaired aluminum wires (5 μm) winding across the 500 μm wide region. Thebroad metal areas on either side of the elements are bonding locationsfor connection to external circuitry. The width of the aluminum elementis 5 μm. The channel in FIG. 5C has identical width and designconfigurations as the heating element lanes in FIG. 5A, and is uniformlyetched 500 μm wide and approximately 20 μm deep.

The heating-element wafer was bonded to a glass wafer containing etchedchannels with the same "Y" format. An aqueous chemical etch ofconcentrated hydrofluoric acid was used to produce channels with definedside walls and uniform depth. The etched channels are defined by achromium/gold mask and are 500 μm wide and approximately 20 μm deep(FIG. 3C). The complementary silicon heater and glass channel waferswere aligned and then bonded with adhesive to form the finished device.

Each heating element used as a temperature sensor is preferably firstcalibrated by measurement of electrical resistance at 22° C. and 65° C.under constant voltage; intermediate temperatures are estimated bylinear interpolation.

II. Channel Treatment

Prior to performing microdroplet motion and biological reactions, thechannels are preferably treated by washing with base, acid, buffer,water and a hydrophilicity-enhancing compound, followed by a relativelyhigh concentration solution of nonspecific protein. In a preferredembodiment, the channels are washed with approximately 100 μl each ofthe following solutions in series: 0.1N NaOH; 0.1N HCl; 10 mM Tris-HCl(pH 8.0), deionized H₂ O, Rain-X Anti-Fog (a hydrophilicity-enhancingcompound commercially available from Unelko Corp., Scottsdale, Ariz.),and 500 μg/μl bovine serum albumin (non-specific protein commerciallyavailable in restriction enzyme grade from GIBCO-BRL). The wafer wasplaced on a stereoscope stage (Olympus SZ1145), and the contact pads forthe heating elements were connected to a regulated power supply. Heatingoccurred by passing approximately 30 volts through the element in shortpulses and observing the movement rate of the droplets. A detectablereduction in droplet volume from evaporation was noted in eachexperiment, usually of less than 30%. Droplet movement was recorded witha Hamamatsu video camera on videotape.

III. Component Design

The present invention contemplates one or more gel electrophoresismodules as a component of the microscale device. Theoretical andempirical research has indicated that reducing the thickness of theelectrophoresis channel leads to improved resolution. Thinner gelsdissipate heat more readily and allow higher voltages to be used, withconcomitant improvements in separation. The position and width of theelectrophoresis detector are also critical to the ultimate resolution ofthe electrophoresis system. A micromachined electronic detector, such asa photodiode, placed in the underlying silicon substrate can be lessthan one micron from the gel matrix and can have a width of 5 microns orless. Since the gel length required for the resolution of two migratingbands is proportional to the resolution of the detector, theincorporation of micron-width electronic detectors can reduce the totalgel length required for standard genotyping by at least an order ofmagnitude.

To demonstrate that standard gel electrophoresis can operate inmicron-diameter channels, modules were fabricated using etched glasschannels identical to FIG. 4B and fluorescent-labeled DNA (YOYOintercalating dye). Polyacrylamide gel electrophoresis of a complex DNAmixture is shown in FIG. 6A in a channel 500 μm wide and 20 μm deep. Theelectrophoresis was performed with the positive electrode to the rightand the DNA sample applied at the left. The white vertical line is thegel-to-buffer interface. The DNA sample (BluescriptKS digested withMspI) is labeled with intercalating UV-fluorescent dye (YOYO-1) and isvisualized under incandescent light. Separation of the component bandsis clearly visible less than 300 μm from the buffer reservoir-to-gelinterface. The high resolution of the detector (in this case, amicroscope) allowed the use of an unusually short gel, resolving severalclosely eluting bands.

The present invention contemplates an electrophoresis unit thatintegrates a micromachined channel and an electronic DNA detector. Thechannel is constructed using a sacrificial etch process on a singlesilicon wafer rather than the bonded surface-etch method describedearlier. In the sacrificial etch technique, the channel configuration ispatterned by depositing on the wafer surface an etch-sensitive material(phosphosilicate glass, SiO₂.P_(x)) with a thickness equivalent to thedesired channel height. A triple-layer overlay of plasma-enhancedchemical vapor deposited silicon nitride, undoped polycrystallinesilicon, and silicon nitride (Si_(x) N_(y) /polySi/Si_(x) N_(y))completely covers the sacrificial material with the exception of smallaccess holes on the top or sides. A selective liquid etch removes thesacrificial layer material, but not the overlay or the underlyingsubstrate. The sacrificial etch technique results in a complete channelbeing formed directly on the substrate containing the electroniccomponents (FIGS. 4C and 4D). The 3 μm deep channel has two bufferreservoirs on either end with integral phosphorus-doped polycrystallinesilicon electrodes. The channel height formed by this technique (˜3 μm)is considerably smaller than the height of the bonded structures due tothe limitations of the sacrificial layer deposition and the strength ofthe overlying layer. Note that, for these channel dimensions, liquiddrops would have volumes on the order of picoliters.

FIG. 6B is photomicrograph of a set of four doped-diffusion dioderadiation detector elements fabricated on a silicon wafer. For eachelement, the three parallel dark lines define the diffusion regions ofthe central the detector flanked by the guard ring shielding electrodes.The diffusion regions are approximately 300 μm long and 4 μm wide.

A radiation detector, consisting of a 10 μm wide "p-n"-type diode with a5 μm wide guard ring around the outer edge, is fashioned directly intothe silicon substrate underneath the channel. In this implementation, anintegral radiation detector was chosen because of (i) high sensitivity(a single decay event), (ii) small aperture dimensions, and (iii)well-know fabrication and response characteristics. On thiselectrophoresis system, a 1 cm long, 3 μm thick gel is able to performas separation on a 80 and a 300 base-pair fragment of DNA. It should benoted that this diode, although currently configured for high-energybeta particle detection, can also operate as a photon detector. Withproper wavelength filters and light sources, detection of fluorescenceemission may be accommodated with a similar device.

Radiation detectors were prepared as follows. A 2001/2-cm, <100>, floatzone, boron-doped, p-type silicon wafer was used as a substrate.Diffused layers of phosphorus (5×10¹⁴ cm⁻²) and boron (1×10¹⁵ cm⁻²) wereion-implanted onto the sample in lithographically-defined regions;thermal silicon oxide was grown (0.2 μm at 900° C.) over the wafer; andcontact holes were etched to the diffusion layer using bufferedhydrofluoric acid solution (5:1). A 3.3 μm layer of Microposit 1400-37photoresist was patterned to define the metal pads; 50 nm chromiumfollowed by 400 nm gold was evaporated over the resist; and themetallization lifted off the regions retaining the resist. A layer ofMicroposit 1813 photoresist was applied across the wafer and baked for110° C. for 30 minutes to form an aqueous solution barrier. Radioactivephosphorus (³² P) decay events could be detected using a sample oflabeled DNA in PCR reaction buffer placed on the photoresist layer. Thedetector was connected to a charge-sensitive preamplifier (EV-Products550A), followed by a linear shaping amplifier and a standardoscilloscope.

FIG. 6C shows an oscilloscope trace of output from the radiationdetector showing individual decay events from ³² P-labeled DNA. Theaqueous DNA sample was placed directly on the detector and sampled for30 seconds. The screen is displaying a vertical scale of 0.5V/divisionand horizontal scale of 20 μsec/division.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms); L (liters);ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters);μm (micrometers); nm (nanometers); °C. (degrees Centigrade); Ci(Curies); MW (molecular weight); OD (optical density); EDTA(ethylenediamine-tetracetic acid); PAGE (polyacrylamide gelelectrophoresis); UV (ultraviolet); V (volts); W (watts); mA(milliamps); bp (base pair); CPM (counts per minute).

EXAMPLE 1

This example describes approaches to the problem of forming a moisturebarrier over electrical elements of the microscale device. Initialprototypes employed 5000 angstroms of aluminum and covered it with PECVDSiO_(x). Upon testing, it was determined that the liquids werepenetrating this later and destroying the aluminum heating elements.

Without clear evidence what was causing this problem, it washypothesized that the step height of the aluminum was causing cracks inthe passivation layer (the oxide). In order to alleviate the crackingproblem, a layer of Si_(x) N_(y) was tried between two layers ofSiO_(x), with the thought that the additional thickness would overcomethe cracking caused by the step height. It did not.

As a follow-up approach, a thinner layer (500 angstroms) of aluminum wastried. This gave 1/10th the step height of the original prototypedevices. On top of this aluminum, a triple layer of SiO_(x), Si_(x)N_(y), and SiO_(x) was employed. Moreover, the process for making theSi_(x) N_(y) layer was changed to one which would give a more denselayer. This appeared to solve the problem. However, the thinner layer ofaluminum created a higher resistance which was not acceptable. It wasdetermined that one needed a way to generate thicker layers of aluminumfor lower resistance, yet keep the surface relatively smooth (planar).An etch back process was used (now called "the inlay process") toaccomplish the task. By etching back into a layer of SiO_(x) depositingaluminum in the resulting cavity, then stripping the resist mask, asurface was obtained with a step height low enough to prevent crackingof the passivation layers.

It was also discovered that the metal bonding pads were not adheringwell to the initial PECVD SiO_(x) layer. To overcome the problem, theprocess was modified by using a wet thermal SiO₂ layer.

EXAMPLE 2

This example describes approaches to enhancing droplet motion by surfacetreatment. In this regard, the principle of using surface tension tocause droplets to move may be applied to either hydrophilic orhydrophobic surfaces. Glass, for instance, is naturally hydrophilic witha near zero contact angle with water. Because the oxide coating of thepresent invention is made principally of the same material as glass, itwas expected that the devices would also exhibit near zero angles. Itwas discovered, however, that the actual construction materials hadcontact angles far from zero, thus enhancing the effects of contactangle hysteresis (discussed in greater detail in Example 3). Forinstance, water gave a contact angle (static) of ˜42° on polyamide, ˜41°on SiO₂ (major component of most glasses), ˜62° on silicone spray. Toenhance the surface effectiveness, several treatment processes for bothhydrophilic and hydrophobic surfaces were tried, as described below.

To improve the hydrophilicity of a surface, several cleaning procedureswere tried. It has been reported that surface contamination and/orroughness can reduce the hydrophilicity of surfaces. Therefore, a highconcentration chromic acid cleaning, a high concentration sulfuric acidcleaning, a baking procedure (to 600° C. for 8 hrs. to burn offcontaminates), and surface coatings were tried. The acid cleaningprocedures were not as effective as the baking procedure; however,neither proved to be compatible with the devices since the concentratedacids would attack the aluminum pads and the high temperature could pealthe aluminum (melting pt. ˜660° C.) or break the adhesive bond betweenthe heater chip and the channel.

Rain-X antifog (commercially available) as a treatment was observed towork. This is a surface treatment which makes surfaces hydrophilic.Although, the resulting surfaces may not be 0°, by using this coatingthe entire surface gets treated giving a uniform surface for thedroplet. Experimentally, it was found that Rain-X antifog treatmentsgreatly enhanced droplet motion experiments using heat. Another suchtreatment which was tested but which did not work was a material calledSilWet. This material is used in the agriculture industry for enhancingthe wetting of plants with agricultural sprays.

To obtain hydrophobic surfaces, the present inventors tried coatingcapillaries with Rain-X and silane treatments. Neither of these gaveangles much greater than 90°, therefore, would not work with thismechanism. These treatments would have to have given angles ˜180° to beuseful for hydrophobic studies of motion. Eventually, it was discoveredthat one could apply a teflon coating that was sufficiently hydrophobicto possibly warrant future tests.

EXAMPLE 3

This example describes approaches to droplet motion by heat treatment.As noted previously (above), the contact angle on the advancing end of aliquid droplet in motion (known as the advancing contact angle) isgreater that the that on the receding end (receding contact angle). Inthe case of a hydrophilic surface--such as water on glass--this tends tocreate a back pressure countering attempts at forward motion by heatingthe back side of a droplet. This is best shown by a simple modeldescribing laminar flow through a channel.

Average Flow Through a Circular Channel:

    <v>=-ΔP*[R.sup.2 /(8 μL]

where:

Δ=value at back--value at front end of droplet

ΔP=(1/R)*(ΔG)=pressure difference between droplet ends

ΔG=change in surface tension between the ends of the droplet.

R=channel radius

L=droplet length

μ=viscosity

Also, for water, ΔG=-constant*ΔT, where temperature increases lower thesurface tension of most liquids (constant=0.16 dyn/cm for water).

Therefore:

    <v>=-(ΔG)*(1/R)*[R.sup.2 /(8 μL)]=[-0.16*ΔT*R/(8 μL)]

where:

ΔT=T_(back) -T_(front)

giving:

<v>=[0.16*R/(8 μL)]*(T_(back) -T_(front)).

This expression indicates that any heating on the back end of thedroplet (if the front remains at a lower temperature) will cause theliquid droplet to move. This was not the case experimentally, however.By way of studies using glass capillaries, it was found that there was aminimum temperature difference required to move the droplet. This effectis believed to be the result of contact angle hysteresis (CAH). In CAH,the advancing contact angle is greater than the receding contact angleresulting in a sort of back pressure which must be overcome to achievedroplet movement. CAH occurs when the interface is placed in motion(dynamic angles). To account for this effect, it was included in asteady-state (ID) model for flow. For instance, if the advancing angleis 36° and the receding angle is 29° (with the front of the dropletbeing 25° C.), then the back of the droplet would need to be heated to˜60° C. for a 1 mm long droplet in a 20 μm high channel. This is justone example situation.

It was discovered experimentally, however, that the channel dimensionand fluid parameters (other than surface tension) do not affect whetheror not the droplet will move. They do determine the magnitude of motion(if it occurs). What does determine whether motion will occur or not isthe following inequality:

    G.sub.front /G.sub.back >(R.sub.front /R.sub.back)*(cos β.sub.back /cos β.sub.front)

where:

β=contact angle.

The present calculations suggest that a ˜35° C. difference between thefront and back of a droplet should be sufficient to initiate dropletmotion in a system with advancing angles of 36° and receding angles of29° in a 20 μm high channel. Experimental testing of actual deviceshowever, showed that the front of the droplet heats relatively quicklythus reducing the temperature difference needed for movement between thefront and the back of the droplet. This effect required us to use highervoltages to obtain droplet motion. Voltages typically in the range of˜30° Volts were found to be required to obtain motion. Furtherexperiments showed that the resulting temperature difference was ˜40° C.between the front and back of the droplet thus corroborating the initialdetermination of the requirements.

Discrete droplet motion in a micromachined channel structure usingthermal gradients is demonstrated in the videorecorded images of FIG. 4.The device consists of a series of aluminum heaters inlaid on a planarsilicon dioxide substrate (similar to the structure shown in FIG. 2) andbonded by glue to a wet-etched glass channel (20 μm depth, 500 μmwidth). Liquid samples were manually loaded into the two channels on theleft using a micropipette. Heating the left interface of each dropletpropels it toward the intersection of the channels. At the intersection,the droplets meet and join to form a single larger droplet. Note that,since the channel cross-section is 20 μm×500 μm, the volume of each ofthese droplets can be calculated from their lengths and is approximately50 nanoliters.

The heaters along the entire surface of the channel shown in FIG. 4allow it to be used as a thermal reaction chamber in addition to adroplet-motion device. The upper droplet in the figure contains a DNAsample, while the lower contains a restriction digest enzyme (TaqI) anddigestion buffer. Following sample merging, the combined droplet wasmaintained at 65° C. for 30 minutes using the integral heaters andtemperature sensors. The completed enzymatic reaction was confirmed byexpressing the droplet from the right end of the channel and loading itonto a capillary gel electrophoresis system with a laser-inducedfluorescence detector. The chromatogram produced by the silicon-devicesample was similar to chromatograms generated from DNA digests runs in astandard polypropylene microreaction vessel (not shown).

EXAMPLE 4

This example describes various approaches for bonding channels to thesubstrate which contains circuitry for heating and temperature sensingof the device of the present invention (see discussion of two-partconstruction, above). First attempts involved Polyamide; regularpolyamide was unsatisfactory in that it was found the two pieces wouldnot stick together.

Follow-up attempts involved a photo-definable Polyamide. This produced asticky surface, but would not give a perfect seal along the channel. Itwas discovered that the solvents released during the final bakingprocess were causing pockets in the polyamide layer. An adhesion layerwas needed which would seal by `curing` and not release solvents.

Several different epoxies and glues were investigated, as listed below.

    ______________________________________                                        Adhesive  Form    Dries   Texture                                                                              Comments                                     ______________________________________                                         1. Dymax UV  Gel     Clear Rubbery                                                                              Cures on UV                                   Glue    exposure.                                                             2. Carter's Goo Yellow/ Rubbery Dries quickly and                             Rubber  Clear  stringy when thinned.                                          Cement                                                                        3. Borden's Liquid Clear Hard Thin, dries on first                            Krazy Glue    contact.                                                        4. UHU Bond- Gel/ Clear Hard Dries quickly and                                All Goo   stringy when thin.                                                  5. Dennison Paste Clear Hard Will not flow on                                 Permanent    applying.                                                        Glue Stick                                                                    6. Elmer's Thick White Hard Slow drying.                                      Glue-All Liquid                                                               (Borden)                                                                      7. Liquid Nails Thin Wood- Hard Thick, dries quickly                           Paste like  when thinned.                                                    8. Devcon 5- Gel Yellow/ Hard Thick, cures on about                           Minute  Clear  5 min.                                                         Epoxy                                                                         9. Scotch Tape Clear Rubbery Tape.                                            Double-Stick                                                                  Tape                                                                         10. Dow Corn- Thick Frosty Soft Seals but does not                             ing High Gel   bond.                                                          Vacuum                                                                        Grease                                                                       11. Nujol Miner- Liquid Clear Runny Neither seals (doesn't                     al Oil     spread on glass) nor                                               (Perkin    bonds.                                                             Elmer)                                                                       12. Household Gel/ Clear Rubbery Contact cement which                          Goop Goo   dries stringy.                                                    13. Permatex Gel/ Yellow/ Rubbery Dries quickly and                            Weather Goo Clear  stringy when thinned.                                      Strip Cement                                                                 14. Thick Gel Gel Clear Hard Does not cure on                                  Super Glue    contact but does                                                    quickly.                                                                 15. DAP Weld- Goo Orange/ Rubbery Contact cement which                         wood Con-  Clear  gets stringy when                                           tact Cement    thinned.                                                      16. Scotch (3M) Thin Yellow/ Rubbery Spray. `Gooey` but                        Photo Mount Goo Clear  not stringy.                                           Spray                                                                         Adhesive                                                                     17. Silicone Liquid Clear Smooth Spray. Dries to thin,                         Resin (spray)    clear, and sealed                                            Lacquer (GC    coating.                                                       Electronics)                                                               ______________________________________                                    

A preferred glue was a UV cured glue, although the process of applyingthe UV glue is tedious and requires some practice to avoid putting theglue in places where it does not belong, e.g., in the channels.

Hydroxide bonding and screen printing of bonding substances was alsoattempted. Another option was glass tape, but the high temperaturesrequired to melt the tape appeared to be too high for the presentdevices.

EXAMPLE 5

This example describes a nucleic acid amplification reaction on asilicon-based substrate. The established DNA biochemistry steps for PCRoccur within physiological conditions of ionic strength, temperature,and pH. Thus, the reaction chamber components have design limitations inthat there must be compatibility with the DNA, enzymes and otherreagents in solution.

To assess biocompatability, components were added to a standard PCRreaction. The results (see FIG. 7) indicated that crystalline siliconmay not be the ideal material for biological compatibility. Given theseresults, it may be desirable to modify the surface of the micromachinedsilicon substrate with adsorbed surface agents, covalently bondedpolymers, or a deposited silicon oxide layer.

To form a biologically compatible heating element, the present inventorsbegan by coating a standard silicon wafer with a 0.5 μm layer of silicondioxide. Next, a 0.3 μm deep, 500 μm wide channel was etched into thesilicon oxide and gold or aluminum was deposited (0.3 μm thick). Thisinlay process results in a relatively planar surface (FIG. 2A) andprovides a base for deposition of a water-impermeable layer. Theimpermeable layer is made by a sequence of three plasma enhanced vapordepositions: silicon oxide (SiO_(x)), silicon nitride (Si_(x) N_(y)),and silicon oxide (SiO_(x)). Since the materials are deposited from thevapor phase the precise stoichiometries are not known. A thin metalheater design was used for this device rather than the doped-siliconresistive heaters previously demonstrated for micromachined PCR reactionchambers, since the narrow metal inlay allows viewing of the liquidsample through a transparent underlying substrate, such as glass orquartz. Also, the use of several independent heating elements permits asmall number to operate as highly accurate resistive temperaturesensors, while the majority of elements are functioning as heaters.

A device fabricated with metal resistive heaters and oxide/nitride/oxidecoating was tested for biological compatibility and temperature controlby using PCR amplification of a known DNA template sample. The reactionwas carried out on the planar device using twenty microliters of PCRreaction mix covered with mineral oil to prevent evaporation. Thereaction mixture was cycled through a standard 35-cycle PCR temperaturecycling regime using the integral temperature sensors linked to aprogrammable controller. Since the reaction volume was significantlylarger than intended for the original heater design, a polypropylenering was cemented to the heater surface to serve as a sample containmentchamber. In all test cases, the presence of amplified reaction productsindicated that the silicon dioxide surface and the heater design did notinhibit the reaction. Parallel amplification experiments performed on acommercial PCR thermocycler gave similar results. A series of PCRcompatibility tests indicated that the reaction on the device is verysensitive to controller settings and to the final surface material incontact with the sample (not shown).

From the above it should be evident that the present invention can beadapted for high-volume projects, such as genotyping. The microdroplettransport avoids the current inefficiencies in liquid handling andmixing of reagents. Moreover, the devices are not limited by the natureof the reactions, including biological reactions.

We claim:
 1. A method for merging microdroplets comprising:a) providingfirst and second liquid microdroplets each having volumes between 1 and100 nanoliters, a liquid microdroplet delivering means, and a device,said device comprising:i) a housing comprised of silicon, ii) first andsecond microdroplet transport channels etched in said silicon andconnecting to form a third transport channel containing a reactionregion, said first, second and third transport channels the channelshaving a depth between 0.35 and 50 μm and a width between 50 and 1000μm, iii) a microdroplet receiving means in liquid communication withsaid reaction region via said transport channels, and iv) microdropletflow-directing means comprising a series of heating elements arrayedalong said first, second and third transport channels; b) deliveringsaid first liquid microdroplet via said microdroplet delivering means tosaid first transport channel; c) delivering said second liquidmicrodroplet via said microdroplet delivering means to said secondtransport channel; and d) conveying said microdroplets in said transportchannels to said reaction region in said third transport channel viasaid microdroplet flow-directing means by differential heating of saidmicrodroplets by said heating elements, thereby merging said first andsecond microdroplets to create a merged microdroplet.
 2. The method ofclaim 1 wherein, prior to said conveying of step d), said transportchannels are treated with a hydrophilicity-enhancing compound.
 3. Themethod of claim 1 wherein said first microdroplet comprises nucleicacid.
 4. The method of claim 3 wherein said second microdropletcomprises a nuclease capable of acting on said nucleic acid.
 5. Themethod of claim 1 further comprising, after step d) reversing the flowdirection of said merged microdroplet so as to increase the mixing ofsaid first and second microdroplets.
 6. A method for movingmicrodroplets, comprising:a) providing a liquid microdroplet disposedwithin a microdroplet transport channel etched in silicon, said channelin liquid communication with a reaction region and separated by a liquidbarrier from a microdroplet flow-directing means, said flow-directingmeans comprising a series of heating elements arrayed along saidtransport channel and disposed within said silicon; and b) conveyingsaid microdroplet in said transport channel via said microdropletflow-directing means by differential heating of said microdroplet bysaid heating elements.
 7. The method of claim 6 wherein said heatingelements are comprised of aluminum.
 8. The method of claim 6 wherein,prior to said conveying of step b), said transport channel is treatedwith a hydrophilicity-enhancing compound.
 9. The method of claim 6wherein said liquid barrier comprises a first silicon oxide layer, asilicon nitride layer, and a second silicon oxide layer.
 10. The methodof claim 6 further comprising, after step b), initiating a reaction in areaction region into which said microdroplet is conveyed.
 11. The methodof claim 6 wherein said microdroplet comprises a biological sample. 12.The method of claim 11 wherein said sample comprises nucleic acid.